Direct Electrochemistry of Immobilized Human Cytochrome P450 2E1

Fantuzzi, Andrea; Fairhead, M.; Gilardi, Gianfranco

doi:10.1021/ja049855s

Cited by 130 publications

(119 citation statements)

References 16 publications

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“…[15][16][17][18][19][20][21][22][23][24] Particular interest has been focused on the improvement of the electronic coupling of the enzyme with the electrode surface and of its catalytic efficiency. In a fully coupled system reducing equivalents and molecular oxygen consumed are stoichiometrically equivalent to the product formed.…”

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confidence: 99%

Electrochemical Detection of Human Cytochrome P450 2A6 Inhibition: A Step toward Reducing Dependence on Smoking

et al. 2014

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Here the "Molecular Lego" approach was used to achieve the first reported electrochemical signal of human CYP2A6 and to improve its catalytic efficiency on electrode surfaces. The enzyme was fused at the genetic level to flavodoxin from Desulfovibrio vulgaris(FLD) to create the chimeric CYP2A6-FLD. Electrochemical characterization by cyclic voltammetry shows clearly defined redox transitions of the haem domain in both CYP2A6 and CYP2A6-FLD.Electrocatalysis experiments using coumarin as substrate followed by fluorimetric quantification of the product were performed with immobilised CYP2A6 and CYP2A6-FLD. Comparison of the kinetic parameters showed that coumarin catalysis was carried out with a higher efficiency by the immobilised CYP2A6-FLD, with a calculated kcat value significantly higher (P<0.005) than that of CYP2A6, while the affinity for the substrate (KM) remained unaltered. The chimeric system was also successfully used to demonstrate the inhibition of the electrochemical activity of the immobilised CYP2A6-FLD, towards both coumarin and nicotine substrates, by tranylcypromine, a potent and selective CYP2A6 inhibitor.This work shows that CYP2A6 turnover efficiency is improved when the protein is linked to FLD redox module and this strategy can be utilized for the development of new clinically relevant biotechnological approaches suitable for deciphering the metabolic implications of CYP2A6 polymorphism and for the screening of CYP2A6 substrates and inhibitors.

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confidence: 99%

Electrochemical Detection of Human Cytochrome P450 2A6 Inhibition: A Step toward Reducing Dependence on Smoking

et al. 2014

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“…For example, alkane-thiol or other thiol-terminated chains covalently bonded to a noble-metal surface and functionalized at the terminus with a group that binds to a specific site of the protein, which have been used to control enzyme binding orientation (Collinson and Bowden, 1992;Pardo-Yissar et al, 2000;Chen et al, 2002). CYP2E1 has been bonded through thiols, both directly or via a cysteine-maleimide linker, to a gold surface although enzyme orientation remained ambiguous (Fantuzzi et al, 2004). Bonding to the N terminus of enzymes provides a more specific alternative than bonding to surface cystines that can result in undesirable orientations of the enzyme.…”

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confidence: 99%

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Yang¹,

Kabulski²,

Wollenberg³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Cytochrome P450 (P450) enzymes typically require the presence of at least cytochrome P450 reductase (CPR) and NADPH to carry out the metabolism of xenobiotics. To address whether the need for redox transfer proteins and the NADPH cofactor protein could be obviated, CYP2C9 was bonded to a gold electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer (SAM) through which a current could be applied. Cyclic voltammetry demonstrated direct electrochemistry of the CYP2C9 enzyme bonded to the electrode and fast electron transfer between the heme iron and the gold electrode. To determine whether this system could metabolize warfarin analogous to microsomal or expressed enzyme systems containing CYP2C9, warfarin was incubated with the CYP2C9-SAM-gold electrode and a controlled potential was applied.The expected 7-hydroxywarfarin metabolite was observed, analogous to expressed CYP2C9 systems, wherein this is the predominant metabolite. Current-concentration data generated with increasing concentrations of warfarin were used to determine the MichaelisMenten constant (K m ) for the hydroxylation of warfarin (3 M), which is in good agreement with previous literature regarding K m values for this reaction. In summary, the CYP2C9-SAM-gold electrode system was able to carry out the metabolism of warfarin only after application of an electrical potential, but in the absence of either CPR or NADPH. Furthermore, this system may provide a unique platform for both studying P450 enzyme electrochemistry and as a bioreactor to produce metabolites without the need for expensive redox transfer proteins and cofactors.Cytochrome P450 (P450) enzymes are important for drug metabolism in humans, accounting for ϳ75% metabolism of drugs (Williams et al., 2004). Numerous factors affect the P450 metabolism rate and the resulting metabolite structure including electron transfer, protein-protein interactions, concentration and structure of the substrate, and the source and specific means whereby the P450 was prepared (Guengerich, 2007). In practice, it is difficult to isolate the individual contributions of these factors because they are often interdependent. Thus, it is of interest to devise model platforms that can be used to independently control these parameters to monitor their respective effects on metabolism.P450 catalysis requires a constant supply of NADPH as the electron source and cytochrome P450 reductase (CPR) to deliver the electrons.In attempts to obviate the requirement of these redox partners/cofactors for catalysis, P450 enzymes have been immobilized on electrodes so that the electrode supplies electrons to drive the P450 catalytic cycle (Estabrook et al., 1996;Reipa et al., 1997;Gilardi et al., 2002) with effective electrical communication between the electrode and the enzyme being critical (Yang et al., 2008). Direct adsorption of enzymes on bare electrodes, such as gold, platinum, and graphite, results in diminished biocatalytic activity (Habermüller et al., 2000;Joseph et al., 2003;Shumy...

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“…Investigations of electrochemical reduction of P450 have led to clever techniques for effecting electron transfer (ET). These methods include confining the protein within surfactant 2,3 or polyelectrolyte 4,5 films, modifying the electrode surface covalently (mercaptans on gold 6 ) or through adsorption (clay on carbon 7 ) and modifying the enzyme with molecular electronic relays. 8 We are working on electrochemical methods for reduction of the fatty acid hydroxylase flavocytochrome P450 from Bacillus megaterium (BM3).…”

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Reduction of Dioxygen Catalyzed by Pyrene-Wired Heme Domain Cytochrome P450 BM3 Electrodes

et al. 2004

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Direct Electrochemistry of Immobilized Human Cytochrome P450 2E1

Cited by 130 publications

References 16 publications

Electrochemical Detection of Human Cytochrome P450 2A6 Inhibition: A Step toward Reducing Dependence on Smoking

Electrochemical Detection of Human Cytochrome P450 2A6 Inhibition: A Step toward Reducing Dependence on Smoking

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Reduction of Dioxygen Catalyzed by Pyrene-Wired Heme Domain Cytochrome P450 BM3 Electrodes

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